The in vitro screening for acetylcholinesterase inhibition and antioxidant activity of medicinal plants from Portugal.

Essential oil, ethanolic extract and decoction of 10 plant species from interior Portugal were analyzed for their activity towards acetylcholinesterase (AChE) enzyme and their antioxidant activity. Of these, Melissa officinalis, Paronychia argentea, Sanguisorba minor, Hypericum undulatum and Malva silvestris are used in herbal medicine, Laurus nobilis and Mentha suaveolens as condiments, and Salvia officinalis, Lavandula angustifolia and Lavandula pedunculata also as aromatics. Melissa officinalis and Mentha suaveolens showed AChE inhibitory capacity higher then 50% in the essential oil fraction. Laurus nobilis, Hypericum undulatum, and Sanguisorba minor showed a high inhibition value of AChE in the ethanolic fraction, 64% (1 mg ml(-1)) 68% (0.5 mg ml(-1)), and 78% (1 mg ml(-1)), respectively. Higher values of AChE inhibitory activity were found using decoctions of Lavandula pedunculata, Mentha suaveolens and Hypericum undulatum, 68, 69 and 82% (at a concentration of 5mg dry plant ml(-1) of assay), respectively. The free radical scavenger activity was higher for the polar extracts. In the water extracts most of the plants showed values around 90%. When antioxidant activity was measured with the beta-carotene-linoleic acid assay high activity (65-95%) was also found in the water extracts. Hypericum undulatum, Melissa officinalis and Laurus nobilis showed both high AChE inhibitory capacity and antioxidant activity.

Dipeptide synthesis and separation in a reversed micellar membrane reactor.

The synthesis of dipeptide AcPheLeuNH2 catalyzed by alpha-chymotrypsin encapsulated in TTAB/octanol/heptane reversed micelles was investigated in a tubular ceramic membrane reactor, operated in a batch mode. The reaction medium conditions (TTAB concentration, buffer molarity, and pH) were optimized using a factorial design in order to achieve maximum synthesis rates. Hydrated reversed micelles permeated through the membrane together with the substrate ester, dipeptide, and by-products. However, as a result of the low solubility of the peptide in the reaction medium, selective precipitation occurred, thus enabling the complete retention of the solid product by the ultrafiltration membrane and therefore an integration of a separation step in the biotransformation process. In spite of the continuous accumulation of solids inside the reactor, constant permeation flow rates could be maintained throughout the operation. The influence of alpha-chymotrypsin, TTAB, and water concentration on the kinetics and mass transfer of the system was also investigated. The behavior of the system during a continuous experiment was also evaluated.

Application of factorial design to the optimization of peroxidase activity in reverse micelles of bis(2-ethylhexyl)sodium sulfosuccinate/ isooctane.

Two cationic peroxidases isolated from Vaccinium myrtillus were encapsulated in reverse micelles of bis(2-ethylhexyl)sodium sulfosuccinate/isooctane. By using a central composite design, some relevant parameters for the enzymatic activity, such as surfactant and water concentration, pH, and buffer molarity, were analyzed. With the results obtained from this experimental planning, the response surface curves were established. The maximum specific activity obtained (0.19 mM/min. mM of enzyme) was approximately the same for both peroxidases, but the experimental conditions under which this value was attained differed considerably.

Acetylcholinesterase inhibition, antioxidant activity and toxicity of Peumus boldus water extracts on HeLa and Caco-2 cell lines.

This work aimed to study the inhibition on acetylcholinesterase activity (AChE), the antioxidant activity and the toxicity towards Caco-2 and HeLa cells of aqueous extracts of Peumus Boldus. An IC(50) value of 0.93 mg/mL, for AChE inhibition, and EC(50) of 18.7 µg/mL, for the antioxidant activity, was determined. This activity can be attributed to glycosylated flavonoid derivatives detected, which were the main compounds, although boldine and other aporphine derivatives were also present. No changes in the chemical composition or the biochemical activities were found after gastrointestinal digestion. Toxicity of P. boldus decoction gave an IC(50) value 0.66 mg/mL for HeLa cells, which caused significant changes in the cell proteome profile.

Kinetic properties of wild-type and altered recombinant amidases by the use of ion-selective electrode assay method.

A novel assay method was investigated for wild-type and recombinant mutant amidases (EC 3.5.1.4) from Pseudomonas aeruginosa by ammonium ion-selective electrode (ISE). The initial velocity is proportional to the enzyme concentration by using the wild-type enzyme. The specific activities of the purified amidase were found to be 88.2 and 104.2 U mg protein(-1) for the linked assay and ISE methods, respectively. The kinetic constants--Vmax, Km, and Kcat--determined by Michaelis-Menten plot were 101.13 U mg protein(-1), 1.12x10(-2) M, and 64.04 s(-1), respectively, for acrylamide as the substrate. On the other hand, the lower limit of detection and range of linearity of enzyme concentration were found to be 10.8 and 10.8 to 500 ng, respectively, for the linked assay method and 15.0 and 15.0 to 15,000 ng, respectively, for the ISE method. Hydroxylamine was found to act as an uncompetitive activator of hydrolysis reaction catalyzed by amidase given that there is an increase in Vmax and Km when acetamide was used as the substrate. However, the effect of hydroxylamine on the hydrolysis reaction was dependent on the type of amidase and substrate involved in the reaction mixture. The degrees of activation (epsilon(a)) of the wild-type and mutant (T103I and C91A) enzymes were found to be 2.54, 12.63, and 4.33, respectively, for acetamide as the substrate. However, hydroxylamine did not activate the reaction catalyzed by wild-type and altered (C91A and W138G) amidases by using acrylamide and acetamide, respectively, as the substrate. The activating effect of hydroxylamine on the hydrolysis of acetamide, acrylamide, and p-nitrophenylacetamide can be explained by the fact that additional formation of ammonium ions occurred due to the transferase activity of amidases. However, the activating effect of hydroxylamine on the hydrolysis of p-nitroacetanilide may be due to a change in conformation of enzyme molecule. Therefore, the use of ISE permitted the study of the kinetic properties of wild-type and mutant amidases because it was possible to measure initial velocity of the enzyme-catalyzed reaction in real time.

Application of Fourier transform infrared spectroscopy for monitoring hydrolysis and synthesis reactions catalyzed by a recombinant amidase.

This study demonstrates the use of Fourier transform infrared (FTIR) spectroscopy for monitoring both synthesis and hydrolysis reactions catalyzed by a recombinant amidase (EC 3.5.1.4) from Pseudomonas aeruginosa. The kinetics of hydrolysis of acetamide, propionamide, butyramide, acrylamide, benzamide, phenylalaninamide, alaninamide, glycinamide, and leucinamide were determined. This revealed that very short-chain substrates displayed higher amidase activity than did branched side-chain or aromatic substrates. In addition, on reducing the polarity and increasing the substrates' bulkiness, a reduction of the amidase affinity for the substrates took place. Using FTIR spectroscopy it was possible to monitor and quantify the synthesis of several hydroxamic acid derivatives and ester hydrolysis products. These products may occur simultaneously in a reaction catalyzed by the amidase. The substrates used for the study of such reactions were ethyl acetate and glycine ethyl ester. Hydroxylamine was the nucleophile substrate used for the synthesis of acetohydroxamate compounds. Results presented in this article demonstrate the usefulness of FTIR spectroscopy as an important tool for understanding the enzyme structure-activity relationship because it provides a simple and rapid real-time assay for the detection and quantification of amidase hydrolysis and synthesis reactions in situ.

Thin layer chromatographic confirmation of aflatoxin M1 extracted from milk.

Aflatoxin M1 can be confirmed directly on a thin layer plate by reacting the toxin with a mixture of reagents containing p-anisaldehyde. This confirmatory procedure requires only 2 elutions in the same direction using 2 different solvents. The mixture containing p-anisaldehyde is overspotted on M1 after the plate has been developed in toluene-ethyl acetate-ethyl ether-formic acid (25 + 35 + 40 + 5). The plate is heated at 110 degrees C for 10 min and then developed in hexane-acetone-chloroform (15 + 50 + 35). The Rf value of the green fluorescent derivative is less than that of the M1 standard. This confirmatory procedure requires only one-dimensional TLC, so several sample extracts and the standard can be run simultaneously. The minimum detectable quantity of aflatoxin M1 on the TLC plate with this test is 0.3 ng. p-Anisaldehyde reagent solution may also be used as a spray reagent for the confirmation of aflatoxin M1. The procedures described were satisfactory for confirming the mycotoxin in spiked samples of powdered and liquid milk.

Rapid thin layer chromatographic determination of aflatoxin M1 in powdered milk.

A method has been developed for the detection of aflatoxin M1 in milk. The toxin is extracted with chloroform, the extract is evaporated, and the residue is partitioned between carbon tetrachloride and an aqueous saline-methanol solution. The toxin is once again extracted with chloroform from the methanol solution and analyzed by thin layer chromatography. The limit of detection of M1 in powdered milk is 0.5 microgram/kg; recoveries of added M1 are about 83%. The limit of detection can be improved to 0.3 microgram/kg if the plate is sprayed with an aqueous solution of H2SO4 after development.

Synthesis of AcPheLeuNH(2) by alpha-chymotrypsin in TTAB reversed micelles: Application of response surface methodology to the optimization of the system.

The influence of the long chain alcohols, hexanol, octanol, and decanol, as cosurfactants of the reverse micellar system of tetradecyltrimethylammonium bromide on the alpha-chymotrypsin-mediated AcPheLeuNH(2) synthesis was studied. The effect of temperature, buffer molarity, pH, and substrate concentration was also evaluated. The enzyme was chemically modified and the effect of this modification upon the enzyme activity was also analyzed. Octanol allowed a higher activity for both enzyme forms. The peptide synthesis/substrate hydrolysis ratio is independent of the long chain alcohol used. The chemical modification decreases the alpha-chymotrypsin activity under the system conditions studied, but increases the initial velocity of peptide synthesis relative to the ester substrate hydrolysis. The response surface methodology was applied to optimize the dipeptide synthesis in the system containing octanol as cosurfactant. (c) 1994 John Wiley & Sons, Inc.

Measuring enzymatic activity of a recombinant amidase using Fourier transform infrared spectroscopy.

A method based on Fourier transform infrared spectroscopy (FT-IR) has been developed for assaying the Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain. The kinetic of acetamide hydrolysis by the enzyme, in aqueous media, was monitored by measuring the intensity of the acetamide amide I band maximum at 1635 cm(-1) as a function of time. A value of 0.5mM(-1) cm(-1) was obtained for the extinction coefficient (epsilon) of acetamide at this frequency. The rate of the hydrolysis was found to be linear with the concentration of the enzyme up to 90 microM. The Michaelis-Menten kinetics parameters V and K(m) were determined as 30.7 U/mg and 4mM, respectively. These results were similar to those obtained using high-performance liquid chromatography analysis of the same hydrolytic reaction catalyzed by amidase either in water or in buffer. This suggests that the precision of the FT-IR method is suitable for the kinetic studies of amidase with the additional advantage of being able to perform a real-time measurement of the enzymatic activity.

Application of empirical design methodologies to the study of the influence of reaction conditions and N-alpha-protecting group structure on the enzymatic X-Phe-Leu-NH(2) dipeptide synthesis in buffer/dimethylformamide solvents systems.

The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH(2) was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-alpha protection choice. Boc and Z were the most reactive N-alpha groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.